Lipid raft membrane compartmentalization and MAGUK-family molecular scaffolds function as key organizers of protein and membrane trafficking, cytoskeletal reorganization and signal transduction in neuronal and epithelial cell junctions. Here we consider a potential role for the Dlgh1 MAGUK family member in organizing the cytoskeleton, lipid rafts and signal transducers at the T cell:APC synaptic junction. During the past funding cycle we demonstrated a requirement for the LckSHS domain in mediating TCR/costimulation induced: synaptic raft clustering; processive and sustained signaling; reduced required duration of TCR engagement; Erk activation; and IL-2 production. We became interested in Dlgh1 as a potential LckSH3 effector because we identified it as a LckSH3 ligand and demonstrated LckSH3: Dlgh1 interactions as essential for Dlgh1 raft microdomain membrane localization. Moreover, we have recently identified WASp, Erk-1 and p38 as additional Dlgh1 ligands and suggest they may function as Dlgh1 (rLck) effectors. In this proposal, we design experiments aimed at assessing a potential role for Dlgh1 in TCR/costimulator induced signal transduction and elucidating the molecular basis of Dlgh1 activity. Furthermore, we consider the possibility that distinct T cell subsets differentially capitalize on Dlgh1 activities to generate synapses uniquely suited for effecting particular functions. To address these issues we capitalize on a three pronged approach involving siRNA mediated Dlgh1 knockdown, Dlgh1 over-expression/re-expression and Dlgh1 gene knockout. We include analysis of Dlgh1 activity in the BI-141 T hybridoma, CD4+ 5CC7 and CD8+ OT-1 TCR transgenic T cells and in developing T cell subsets. We predict that direct comparison of Dlgh1 scaffolds within distinct developing and T effector populations will elucidate novel TCR signal transduction mechanisms and molecular details involved in specializing synapses. Specifically, we propose the following: Arm 1) To investigate potential roles for Dlgh1 in antigen-induced T cell signal transduction, immune synapse assembly, and effector function; Aim 2) To determine the molecular basis of Dlgh1 activity in T cells; and Aim 3) To determine whether developing and effector T cells (differentially) rely on Dlgh1 scaffolding activities. Our studies will likely lead to a better understanding of how TCR signals are regulated to mediate functional outcome, which is essential to the design of therapeutics aimed at predictably modulating particular TCR responses. Indeed, elucidation of molecular mediators of the synaptic organization and signal transduction events will provide novel targets for therapeutics aimed at inhibiting unwanted T cell activation responses including autoimmunity, graft rejection, and T cell transformation. Conversely, facilitators of individual activation events could be used in the design of tumor or other vaccines aimed at potentiating responses against suboptimally presented antigens.